Publications by authors named "Lev P Ovchinnikov"

32 Publications

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

Commun Biol 2021 Mar 19;4(1):359. Epub 2021 Mar 19.

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025, Evry, France.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-021-01862-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979924PMC
March 2021

Y-Box Binding Proteins in mRNP Assembly, Translation, and Stability Control.

Biomolecules 2020 04 11;10(4). Epub 2020 Apr 11.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.

Y-box binding proteins (YB proteins) are DNA/RNA-binding proteins belonging to a large family of proteins with the cold shock domain. Functionally, these proteins are known to be the most diverse, although the literature hardly offers any molecular mechanisms governing their activities in the cell, tissue, or the whole organism. This review describes the involvement of YB proteins in RNA-dependent processes, such as mRNA packaging into mRNPs, mRNA translation, and mRNA stabilization. In addition, recent data on the structural peculiarities of YB proteins underlying their interactions with nucleic acids are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10040591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226217PMC
April 2020

Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus.

Cells 2019 Dec 31;9(1). Epub 2019 Dec 31.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016903PMC
December 2019

Translation of Human β-Actin mRNA is Regulated by mTOR Pathway.

Genes (Basel) 2019 01 29;10(2). Epub 2019 Jan 29.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, 142290 Moscow, Russia.

The mammalian target of rapamycin (mTOR) kinase is a well-known master regulator of growth-dependent gene expression in higher eukaryotes. Translation regulation is an important function of the mTORC1 pathway that controls the synthesis of many ribosomal proteins and translation factors. Housekeeping genes such as () are widely used as negative control genes in studies of growth-dependent translation. Here we demonstrate that translation of both endogenous and reporter mRNA is inhibited in the presence of mTOR kinase inhibitor (Torin1) and under amino acid starvation. Notably, 5'UTR and promoter of are sufficient for the mTOR-dependent translational response, and the degree of mTOR-sensitivity of mRNA translation is cell type-dependent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes10020096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410274PMC
January 2019

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

Nucleic Acids Res 2019 04;47(6):3127-3141

SABNP, University of Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky1303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451097PMC
April 2019

Transportin-1-dependent YB-1 nuclear import.

Biochem Biophys Res Commun 2016 Nov 26;480(4):629-634. Epub 2016 Oct 26.

Institute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., 142290, Pushchino, Moscow Region, Russia. Electronic address:

The DNA/RNA-binding protein YB-1 (Y-box binding protein 1) performs multiple functions both in the cytoplasm and the nucleus of the cell. Generally localized to the cytoplasm, under certain conditions YB-1 is translocated to the nucleus. Here we report for the first time a transport factor that mediates YB-1 nuclear import - transportin-1. The YB-1/transportin-1 complex can be isolated from HeLa cell extract. Nuclear import of YB-1 and its truncated form YB-1 (1-219) in in vitro transport assay was diminished in the presence of a competitor substrate and ceased in the presence of transportin-1 inhibitor M9M. Inhibitors of importin β1 had no effect on YB-1 transport. Furthermore, transport of YB-1 (P201A/Y202A) and YB-1 (1-219) (P201A/Y202A) bearing inactivating mutations in the transportin-1-dependent nuclear localization signal was practically abolished. Together, these results indicate that transportin-1 mediates YB-1 nuclear translocation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2016.10.107DOI Listing
November 2016

Poly(ADP-ribosyl)ation as a new posttranslational modification of YB-1.

Biochimie 2015 Dec 9;119:36-44. Epub 2015 Oct 9.

Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, 630090, Russia; Novosibirsk State University, Novosibirsk, 630090, Russia. Electronic address:

Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions. Therefore, poly(ADP-ribosyl)ation of YB-1 catalyzed with PARP1, can be stimulated by damaged DNA. The observed property of YB-1 underlines its ability to participate in the DNA repair by its involvement in the regulatory cascades of DNA repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2015.10.008DOI Listing
December 2015

mRNA and DNA selection via protein multimerization: YB-1 as a case study.

Nucleic Acids Res 2015 Oct 13;43(19):9457-73. Epub 2015 Aug 13.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université Evry-Val d'Essonne, Evry, 91025 France

Translation is tightly regulated in cells for keeping adequate protein levels, this task being notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of mRNAs to repress or facilitate their translation. To select specific mRNAs, mRNA-binding proteins can strongly bind to specific mRNA sequences/structures. However, many mRNA-binding proteins rather display a weak specificity to short and redundant sequences. Here we examined an alternative mechanism by which mRNA-binding proteins could inhibit the translation of specific mRNAs, using YB-1, a major translation regulator, as a case study. Based on a cooperative binding, YB-1 forms stable homo-multimers on some mRNAs while avoiding other mRNAs. Via such inhomogeneous distribution, YB-1 can selectively inhibit translation of mRNAs on which it has formed stable multimers. This novel mechanistic view on mRNA selection may be shared by other proteins considering the elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we also demonstrate how, by using the same mechanism, YB-1 can form multimers on specific DNA structures, which could provide novel insights into YB-1 nuclear functions in DNA repair and multi-drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627072PMC
October 2015

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1.

J Mol Recognit 2015 Feb 21;28(2):117-23. Epub 2015 Jan 21.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia.

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmr.2435DOI Listing
February 2015

Alternative forms of Y-box binding protein 1 and YB-1 mRNA.

PLoS One 2014 12;9(8):e104513. Epub 2014 Aug 12.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

The multifunctional eukaryotic protein YB-1 (Y-box binding protein 1) plays a role in DNA reparation, transcription regulation, splicing, and mRNA translation, thereby participating in many crucial events in cells. Its effect is dependent mostly on its amount, and hence, on regulation of its synthesis. Published data on regulation of synthesis of YB-1 mediated by its mRNA 5' UTR, and specifically on the 5' UTR length and the presence of TOP-like motifs in this region, are contradictory. Here we report that 5' UTRs of major forms of human, rabbit, and mouse YB-1 mRNAs are about 140 nucleotides long and contain no TOP-like motifs mentioned in the literature. Also, we have found that YB-1 specifically interacts with the 5' UTR of its own mRNA within a region of about 100 nucleotides upstream from the start codon. Apart from YB-1, translation of YB-1 mRNA in a cell free system gives an additional product with an extended N-terminus and lower electrophoretic mobility. The start codon for synthesis of the additional product is AUC at position -(60-58) of the same open reading frame as that for the major product. Also, in the cell there is an alternative YB-1 mRNA with exon 1 replaced by a part of intron 1; YB-1 synthesized in vitro from this mRNA contains, instead of its N-terminal A/P domain, 10-11 amino acids encoded by intron 1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104513PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4130533PMC
April 2015

YB-1 protein: functions and regulation.

Wiley Interdiscip Rev RNA 2014 Jan-Feb;5(1):95-110. Epub 2013 Nov 11.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

The Y-box binding protein 1 (YB-1, YBX1) is a member of the family of DNA- and RNA-binding proteins with an evolutionarily ancient and conserved cold shock domain. It falls into a group of intrinsically disordered proteins that do not follow the classical rule 'one protein-one function' but introduce a novel principle stating that a disordered structure suggests many functions. YB-1 participates in a wide variety of DNA/RNA-dependent events, including DNA reparation, pre-mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. At the cell level, the multiple activities of YB-1 are manifested as its involvement in cell proliferation and differentiation, stress response, and malignant cell transformation. WIREs RNA 2014, 5:95-110. doi: 10.1002/wrna.1200 CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/wrna.1200DOI Listing
July 2014

The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress.

Cell Cycle 2013 Dec 7;12(24):3791-803. Epub 2013 Oct 7.

Institute of Protein Research; Russian Academy of Sciences; Pushchino, Moscow Region, Russian Federation.

The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.26670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905071PMC
December 2013

YB-1 synthesis is regulated by mTOR signaling pathway.

PLoS One 2012 20;7(12):e52527. Epub 2012 Dec 20.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

YB-1 is a eukaryotic protein with numerous intra- and extracellular functions based on its ability to interact with RNA, DNA, and many proteins. In spite of achievements in studying its functions, regulation of YB-1 synthesis in the cell remains poorly understood. In the current study Western and Northern blotting were used to determine the amounts of YB-1 and YB-1 mRNA in rabbit organs and several cell lines. As found, in the majority of studied eukaryotic cells a considerable proportion of YB-1 mRNA was stored in free mRNPs, i.e., was poorly translated. Also, we demonstrated that YB-1 synthesis depended on conditions that determined the rate of cell division. Specific suppression of YB-1 synthesis resulted from inhibition of the mTOR signaling pathway with inhibitor PP242, but not rapamycin. Experiments on reporter constructs showed that dependence of YB-1 mRNA translation on activity of the mTOR signaling pathway was dictated by 5' untranslated regions of this mRNA, irrelatively of the TOP-like sequences at the beginning of 5' UTR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052527PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527543PMC
June 2013

The major mRNP protein YB-1: structural and association properties in solution.

Biochim Biophys Acta 2013 Feb 5;1834(2):559-67. Epub 2012 Dec 5.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2012.11.007DOI Listing
February 2013

Specific PABP effect on translation of YB-1 mRNA is neutralized by polyadenylation through a "mini-loop" at 3' UTR.

RNA Biol 2012 Dec 7;9(12):1473-87. Epub 2012 Nov 7.

Institute of Protein Research; Russian Academy of Sciences; Pushchino, Moscow Region, Russian Federation.

YB-1 is a multifunctional cold shock domain containing protein that is involved virtually in all DNA- and mRNA-dependent cellular events. Its amount is regulated at the level of both transcription and translation. We showed previously that translation of poly A(-) YB-1 mRNA in vitro is selectively controlled by two proteins, YB-1 and PABP, through their specific and competitive binding to a regulatory element (RE) within 3' UTR of this mRNA. Here, we describe effects of these two proteins on translation of poly A(+) as compared with poly A(-) YB-1 mRNA in a rabbit reticulocyte cell-free translation system. We have found that YB-1 inhibits translation of both poly A(+) and poly A(-) YB-1 mRNAs at the same comparatively low YB-1/mRNA ratio. PABP has no positive effect on translation of poly A(+) YB-1 mRNA, although it has a stimulating effect on translation of poly A(-) YB-1 mRNA. A positive PABP effect on translation of poly A(+) YB-1 mRNA arose after removal of a portion of the sequence between RE and the poly(A) tail and disappeared after its replacement by another non-specific sequence of the same length. We also report that the RE fragment forms a complex with the poly(A) fragment in the presence of rabbit reticulocyte lysate (RRL) proteins. For its formation PABP is necessary but not sufficient. These results are in agreement with the proposed model implying formation of a mini-loop at 3' UTR of YB-1 mRNA that includes RE, RRL proteins and the poly(A) tail.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/rna.22711DOI Listing
December 2012

Formation of amyloid-like fibrils by Y-box binding protein 1 (YB-1) is mediated by its cold shock domain and modulated by disordered terminal domains.

PLoS One 2012 8;7(5):e36969. Epub 2012 May 8.

Group of Protein Biosynthesis Regulation, Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia.

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348147PMC
September 2012

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1.

J Mol Recognit 2012 Apr;25(4):224-33

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk, 630090, Russia.

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmr.2182DOI Listing
April 2012

Interplay between Y-box-binding protein 1 (YB-1) and poly(A) binding protein (PABP) in specific regulation of YB-1 mRNA translation.

RNA Biol 2011 Sep-Oct;8(5):883-92. Epub 2011 Jul 26.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

YB-1 is a DNA- and RNA-binding protein that regulates expression of many important genes. Its deficiency or excess may pose threats, including malignant cellular transformation and metastasis, which explains the necessity of strict control over its amount at every level. As we showed previously, the 3' untranslated region (UTR) of YB-1 mRNA contains a regulatory element specifically binding to YB-1 and PABP (PABPC1). Also, we showed that YB-1 selectively inhibits YB-1 mRNA translation, while PABP stimulates it in a poly(A) tail-independent manner. It was suggested that regulation of YB-1 mRNA translation involves competition between PABP and YB-1 for binding to the regulatory element. Here we offer cogent evidence for this model and add novel details to the mechanism of regulation of YB-1 synthesis. In experiments on regulatory element deletion we showed that it is this element that is responsible for a specific effect of YB-1 and PABP on YB-1 mRNA translation. Mutations eliminating only specific YB-1 affinity for this element suppressed the inhibitory effect of YB-1 and concurrently dramatically decreased the PABP stimulating effect. Mutations reducing only specific PABP affinity for this element, as well as spatial separation of the YB-1- and PABP binding sites, did not affect the YB-1 inhibitory action but completely abolished the positive PABP effect. Together, these results unambiguously prove direct inhibitory action of YB-1 on its mRNA translation, while the positive effect of PABP is realized through displacing YB-1 from the regulatory element.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/rna.8.5.16022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256357PMC
January 2013

Novel findings on endoribonuclease activity of proteasomes.

Cell Cycle 2010 Mar 15;9(6):1028. Epub 2010 Mar 15.

Russian Academy of Sciences, Moscow, Russia.

View Article and Find Full Text PDF

Download full-text PDF

Source
March 2010

Role of microtubules in stress granule assembly: microtubule dynamical instability favors the formation of micrometric stress granules in cells.

J Biol Chem 2009 Dec 19;284(52):36569-36580. Epub 2009 Oct 19.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM/Université d'Evry-Val d'Essonne, U829, EA3637, Evry 91025, France. Electronic address:

Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only approximately 10-20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.042879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794772PMC
December 2009

Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition.

Cancer Cell 2009 May;15(5):402-15

Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC V5Z 1L3, Canada.

Increased expression of the transcription/translation regulatory protein Y-box binding protein-1 (YB-1) is associated with cancer aggressiveness, particularly in breast carcinoma. Here we establish that YB-1 levels are elevated in invasive breast cancer cells and correlate with reduced expression of E-cadherin and poor patient survival. Enforced expression of YB-1 in noninvasive breast epithelial cells induced an epithelial-mesenchymal transition (EMT) accompanied by enhanced metastatic potential and reduced proliferation rates. YB-1 directly activates cap-independent translation of messenger RNAs encoding Snail1 and other transcription factors implicated in downregulation of epithelial and growth-related genes and activation of mesenchymal genes. Hence, translational regulation by YB-1 is a restriction point enabling coordinated expression of a network of EMT-inducing transcription factors, likely acting together to promote metastatic spread.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccr.2009.03.017DOI Listing
May 2009

General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation.

EMBO J 2009 Jan 11;28(1):58-68. Epub 2008 Dec 11.

Department of Biochemistry and Goodman Cancer Center, McGill University, Montreal, Quebec, Canada.

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2008.259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633083PMC
January 2009

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules.

BMC Biochem 2008 Sep 15;9:23. Epub 2008 Sep 15.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM/UEVE U829 Evry, 91025 France.

Background: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.

Results: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.

Conclusion: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2091-9-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557009PMC
September 2008

Atomic force microscopy reveals binding of mRNA to microtubules mediated by two major mRNP proteins YB-1 and PABP.

FEBS Lett 2008 Aug 22;582(19):2875-81. Epub 2008 Jul 22.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

A significant fraction of mRNAs is known to be associated in the form of mRNPs with microtubules for active transport. However, little is known about the interaction between mRNPs and microtubules and most of previous works were focused on molecular motor:microtubule interactions. Here, we have identified, via high resolution atomic force microscopy imaging, a significant binding of mRNA to microtubules mediated by two major mRNP proteins, YB-1 and PABP. This interaction with microtubules could be of critical importance for active mRNP traffic and for mRNP granule formation. A similar role may be fulfilled by other cationic mRNA partners.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2008.07.019DOI Listing
August 2008

Y-box binding protein 1: providing a new angle on translational regulation.

Cell Cycle 2006 Jun 1;5(11):1143-7. Epub 2006 Jun 1.

Department of Pathology, Child and Family Research Institute, University of British Columbia, Canada.

Current models of translational regulation are mostly focused on how translational factors engage a messenger mRNA to the ribosome to initiate translation. Since the majority of mRNAs in eukaryotes are translated in a cap-dependent manner, the mRNA 5' cap-binding protein eIF4E was characterized as a key player responsible for the recruitment of mRNAs to the initiation complex. The availability of eIF4E is believed to be especially critical for translational activation of mRNAs with extensive secondary structures in their 5'UTRs, many of which code for labile regulatory proteins essential for cell growth or viability. Surprisingly, little attention is paid to the other side of translational control, e.g., to define mechanisms responsible for translational silencing and storage of the above messages. In this review, we discuss the possibility that eIF4E per se may not be sufficient to release mRNAs from translational block. We found that many growth- and stress-related mRNAs are associated with the translational repressor YB-1, which can compete with the eIF4E-driven translation initiation complex for binding to the capped 5' mRNA terminus. Moreover, the cap-dependent repressor activity of YB-1 appears to be negatively regulated via Akt-mediated phosphorylation of the Ser-102 residue of YB-1. Taken together with recent evidence suggesting that translational activation of growth-related messages is a primary cellular response to activation of Ras-Erk and PI3K-Akt signaling pathways, our data suggest that differential expression of specific mRNA subsets is regulated by the PI3K-Akt pathway and achieved via coordinated activation of the components of translational machinery and inactivation of general translational repressors such as YB-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.5.11.2784DOI Listing
June 2006

Akt-mediated YB-1 phosphorylation activates translation of silent mRNA species.

Mol Cell Biol 2006 Jan;26(1):277-92

Department of Pathology, British Columbia Research Institute for Children's and Women's Health, Vancouver, BC, Canada V5Z 163.

YB-1 is a broad-specificity RNA-binding protein that is involved in regulation of mRNA transcription, splicing, translation, and stability. In both germinal and somatic cells, YB-1 and related proteins are major components of translationally inactive messenger ribonucleoprotein particles (mRNPs) and are mainly responsible for storage of mRNAs in a silent state. However, mechanisms regulating the repressor activity of YB-1 are not well understood. Here we demonstrate that association of YB-1 with the capped 5' terminus of the mRNA is regulated via phosphorylation by the serine/threonine protein kinase Akt. In contrast to its nonphosphorylated form, phosphorylated YB-1 fails to inhibit cap-dependent but not internal ribosome entry site-dependent translation of a reporter mRNA in vitro. We also show that similar to YB-1, Akt is associated with inactive mRNPs and that activated Akt may relieve translational repression of the YB-1-bound mRNAs. Using Affymetrix microarrays, we found that many of the YB-1-associated messages encode stress- and growth-related proteins, raising the intriguing possibility that Akt-mediated YB-1 phosphorylation could, in part, increase production of proteins regulating cell proliferation, oncogenic transformation, and stress response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.26.1.277-292.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317623PMC
January 2006

Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response.

EMBO J 2005 Oct 29;24(20):3602-12. Epub 2005 Sep 29.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA- and RNA-dependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acid-long YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin- and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7600830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276713PMC
October 2005

YB-1 autoregulates translation of its own mRNA at or prior to the step of 40S ribosomal subunit joining.

Mol Cell Biol 2005 Apr;25(8):3317-23

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

YB-1 is a member of the numerous families of proteins with an evolutionary ancient cold-shock domain. It is involved in many DNA- and RNA-dependent events and regulates gene expression at different levels. Previously, we found a regulatory element within the 3' untranslated region (UTR) of YB-1 mRNA that specifically interacted with YB-1 and poly(A)-binding protein (PABP); we also showed that PABP positively affected YB-1 mRNA translation in a poly(A) tail-independent manner (O. V. Skabkina, M. A. Skabkin, N. V. Popova, D. N. Lyabin, L. O. Penalva, and L. P. Ovchinnikov, J. Biol. Chem. 278:18191-18198, 2003). Here, YB-1 is shown to strongly and specifically inhibit its own synthesis at the stage of initiation, with accumulation of its mRNA in the form of free mRNPs. YB-1 and PABP binding sites have been mapped on the YB-1 mRNA regulatory element. These were UCCAG/ACAA for YB-1 and a approximately 50-nucleotide A-rich sequence for PABP that overlapped each other. PABP competes with YB-1 for binding to the YB-1 mRNA regulatory element and restores translational activity of YB-1 mRNA that has been inhibited by YB-1. Thus, YB-1 negatively regulates its own synthesis, presumably by specific interaction with the 3'UTR regulatory element, whereas PABP restores translational activity of YB-1 mRNA by displacing YB-1 from this element.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.25.8.3317-3323.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1069629PMC
April 2005

Structural organization of mRNA complexes with major core mRNP protein YB-1.

Nucleic Acids Res 2004 19;32(18):5621-35. Epub 2004 Oct 19.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.

YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA-YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600-700 nt mRNA segment on its surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkh889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC524299PMC
November 2004

Poly(A)-binding protein positively affects YB-1 mRNA translation through specific interaction with YB-1 mRNA.

J Biol Chem 2003 May 19;278(20):18191-8. Epub 2003 Mar 19.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russian Federation.

The major protein of cytoplasmic mRNPs from rabbit reticulocytes, YB-1, is a member of an ancient family of proteins containing a common structural feature, cold-shock domain. In eukaryotes, this family is represented by multifunctional mRNA/Y-box DNA-binding proteins that control gene expression at different stages. To address possible post-transcriptional regulation of YB-1 gene expression, we examined effects of exogenous 5'- and 3'-untranslatable region-containing fragments of YB-1 mRNA on its translation and stability in a cell-free system. The addition of the 3' mRNA fragment as well as its subfragment I shut off protein synthesis at the initiation stage without affecting mRNA stability. UV cross-linking revealed four proteins (69, 50, 46, and 44 kDa) that specifically interacted with the 3' mRNA fragment; the inhibitory subfragment I bound two of them, 69- and 50-kDa proteins. We have identified these proteins as PABP (poly(A)-binding protein) (69 kDa) and YB-1 (50 kDa) and demonstrated that titrating out of PABP by poly(A) strongly and specifically inhibits YB-1 mRNA cap(+)poly(A)(-) translation in a cell-free system. Thus, PABP is capable of positively affecting YB-1 mRNA translation in a poly(A) tail-independent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M209073200DOI Listing
May 2003